Sc 25778

Protein extract from fibroblasts was separated by electrophoresis on a 4–12% polyacrylamide gel (NuPAGE). Immunoblotting was performed as described using antibodies to SSBP1, (12212-1-AP, Proteintech), mitochondrially encoded cytochrome c oxidase 1 (MT-CO1, ab14705, Abcam), mitochondrially encoded cytochrome c oxidase 2 (MT-CO2, 12C4F12, Abcam), succinate dehydrogenase complex flavoprotein subunit A (SDHA, ab14715, Abcam), ATP synthase subunit alpha (ATP5A, MS604, Mitosciences), translocase of outer membrane 20 (TOMM20, ab56783, Abcam) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-25778, Santa-Cruz) (Pfeffer et al., 2014 (link)). Cellular oxygen consumption rate (OCR) was assayed using the Seahorse XF96 Extracellular Flux Analyser with sequential addition of oligomycin (1 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 1 μM) and rotenone/antimycin (1 μM) to measure basal and maximal respiration. Cell growth was assessed by growing fibroblast cells with high glucose medium versus glucose-free medium supplemented with 5 mM galactose using IncuCyte® Live Cell imager, Essen Bioscience.

Collected tissues were homogenized in cell lysis buffer (Beyotime Institute of Biotechnology), with protease inhibitors (Biotool).The homogenates were centrifugated for 10 min (12,000 g), and protein concentration in the supernatant was measured using the BCA Protein Assay Kit (BIOMIGA). Equal amounts of total protein (40–60 μg) were loaded and separated on 12% SDS-PAGE by electrophoresis (120 V, 90–120 min). Proteins on SDS-PAGE were blotted onto PVDF membranes (100 V, 90–120 min). The membranes were blocked with 5% skim milk dissolved in TBST (20mMTris-HCl,150mMNaCl,0.05%Tween-20) solution for 2 h, and incubated sequentially with primary antibodies in TBST against IL-6 (1:10000, abcamab7737), IL-1β (1:2000, R&D Systems, AF-401-NA), TNF-α (1:1000, abcamab1793), Wnt5a (1:2500, abcamab72583), α-tubulin (1:10000, Proteintech,66031-1-lg) or GAPDH (1:10000, Santa Cruz sc-25778) for 2 h.After washes with TBST3 times for 10 minutes each and the membranes were incubated with goat anti-rabbit IgG (1:30000, abcamab97051) or goat anti-mouse IgG (1:30000 abcamab97023) secondary antibody conjugated with horseradish peroxidase (HRP) in TBST, After washes with TBST3 times for 10 minutes each and the protein bands were visualized by ECL (Advansta). Tubulin and GAPDH were used as loading controls.

Western blotting was performed using the following antibodies: rabbit anti-PKA R1α (ab139695, Abcam, 1:1000), rabbit anti-phospho-PKA substrate (9624, Cell Signaling, 1:1000), rat anti-Nrf2 (MABE1799, Millipore Sigma, 1:1000), rabbit anti-Nqo1 (ab34173, Abcam, 1:1000), mouse anti-p62 (ab56416, Abcam, 1:1000), rabbit anti-phospho-p62 (Ser349, ab211324, Abcam, 1:1000), rabbit anti-Ho-1 (82206, Cell Signaling, 1:1000), rabbit anti-Keap1 (7705, Cell Signaling, 1:1000), rabbit anti-LC3A/B (12741, Cell Signaling, 1:1000), rabbit anti-phospho-p70 S6 Kinase (Thr389, 9234, Cell Signaling, 1:1000), rabbit anti-p70 S6 Kinase (9202, Cell Signaling, 1:1000), rabbit anti-phospho-4E-BP1 (Thr37/46, 2855, Cell Signaling, 1:1000), rabbit anti-4E-BP1 (9644, Cell Signaling, 1:1000), rabbit anti-GAPDH (sc-25778, Santa Cruz Biotechnology, 1:1000), and mouse anti-β-actin (sc-47778, Santa Cruz Biotechnology, 1:1000).

Western Blotting was performed according to the Cold Spring Harbor protocol. Briefly, cells were lysed in a RIPA buffer (Boston BioProducts, MA, USA) using 25-gauge syringes. The same protein amounts were subjected to 4–20% gradient gel (Genscript, NJ, USA), followed by transfer to a polyvinylidene fluoride (PVDF) membrane using wet methods where appropriate. The membranes were blocked in blocking solution (LI-COR, Inc., Lincoln, NE, USA) for 60 min unless otherwise specified, and incubated overnight with a mouse monoclonal anti-HSP70 antibody (1/1000, SMC100, StressMarq Biosciences, BC, Canada), a rabbit polyclonal anti-BAG3 antibody (1/1000, 10599-1-AP, Proteintech, IL, USA), a mouse monoclonal anti-HSP90α antibody (1/1000, SMC147, StressMarq, Biosciences, BC, Canada) and a rabbit polyclonal anti-GAPDH antibody (1/1000, sc-25778, Santa Cruz, CA, USA). The membranes were incubated for 1 h at room temperature with goat anti-rabbit IRDye 800 CW fluorescent secondary antibodies (1/15,000, LI-COR, Inc., Lincoln, NE, USA) and 680 RD fluorescent secondary antibodies (1/15,000, LI-COR, Inc., Lincoln, NE, USA). Blots were washed with Tris buffered saline, 0.1% (w/v) Tween 20 (TBS-T) and visualized with the Odyssey Imaging System (LI-COR, Inc., Lincoln, NE, USA). The quantitative densitometric analysis was performed using Image Studio Lite Ver. 5.2 (LI-COR, Inc., Lincoln, NE, USA).

Cells were incubated for 30 min in ice-cold lysis buffer (150 mM NaCl, 10 mM Tris HCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, 1 mM Na₃VO₄, 1 mM NaF, 5 mM sodium pyrophosphate, and a protease inhibitor cocktail). After centrifugation at 20,000 g for 15 min, supernatants were boiled in 1xSDS loading buffer prior to SDS-PAGE. Proteins were transferred to PVDF membrane and blocked with 5% milk/TBST (10 mM Tris, pH 7.5, 150 mM NaCl, 0.15% Tween 20) for 1 h. Subcellular fractionations were performed using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific, P178833) according to manufacturer’s instructions. Blots were probed with primary antibodies in 5% BSA overnight at 4 °C followed by HRP-conjugated secondary antibodies if required and subsequent ECL detection reagent (Thermo Fischer, Pierce ECL Western Blotting Substrate 32106). The PiT1 antibody was prepared in our lab and is an anti-mouse polyclonal rabbit antibody^{9 (link)}. Antibodies against IκB (Cell Signaling, 4814), p65 (Cell Signaling, 4764), lamin (Cell Signaling, 2032), and GAPDH (Santa Cruz Biotechnology, sc-25778) were purchased.

Aliquots of protein (20 μg) were separated on 6.0% or 15% SDS-polyacrylamide gels and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Tokyo, Japan). The blots were blocked by treatment with 5.0% skimmed milk in Tris-buffered saline containing 0.1% Tween-20, and were then incubated with antibodies. The following antibodies were used: SOD1 [16 (link)], SOD2 [16 (link)], NOS2 (sc-8310, Santa Cruz Biotechnology), cyclooxygenase-2 (COX2) (sc-1746, Santa Cruz Biotechnology), peroxiredoxin (Prx) 1 (Prx1) [19 (link)], Prx2 (LF-PA0091, AbFrontier, Seoul, Korea), Prx3 (LFMA0044, AbFrontier), Prx4 [20 (link)], glutathione peroxidase 1 (Gpx1) [21 (link)] and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-25778, Santa Cruz Biotechnology). After incubation with horseradish peroxidase-conjugated anti-mouse (sc-2005, Santa Cruz Biotechnology) or anti-rabbit (sc-2004, Santa Cruz Biotechnology) secondary antibodies, the immunoreactive bands were detected using an Immobilon western chemiluminescent HRP substrate (Millipore) on an image analyzer (ImageQuant LAS500, GE Healthcare, Hino, Japan). The relative amounts of each protein were quantified using the Image J software [22 (link)].